CA2354475C - Blends of isoflavones and flavones - Google Patents
Blends of isoflavones and flavones Download PDFInfo
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- CA2354475C CA2354475C CA2354475A CA2354475A CA2354475C CA 2354475 C CA2354475 C CA 2354475C CA 2354475 A CA2354475 A CA 2354475A CA 2354475 A CA2354475 A CA 2354475A CA 2354475 C CA2354475 C CA 2354475C
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- quercetin
- isoflavones
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- aglucon
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
- A61K8/602—Glycosides, e.g. rutin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/81—Solanaceae (Potato family), e.g. tobacco, nightshade, tomato, belladonna, capsicum or jimsonweed
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/88—Liliopsida (monocotyledons)
- A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
- A61K36/8962—Allium, e.g. garden onion, leek, garlic or chives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
- A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
- A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
Abstract
Blends of quercetin and isoflavones from the group consisting of genestein, daidzein and glycetin display synergistic effects when applied as anti-inflammatory agent or as skin agent in particular for anti ageing purposes.
Description
F 7 5 51 cp 1 ( V ) CA 02354475 2001-08-O1 BLENDS OF ISOFLAVONES AND FLAVONES
Isoflavones are known as health components that can be applied to prevent or treat many health deficiencies or to achieve certain health effects not directly related with a health deficiency. E.g. these compounds are known to achieve benefits in the women's health area in particular for postmenopausal women. These effects are disclosed in e.g. US 5 498 631; WO 98/ 56373; WO 98/08503; US 5 733 926;
US 5 952 374 and many other references. Health effects that are also attributed to isoflavones include skin effects and anti-inflammatory effects.
Although for a few of these effects some experimental support can be found in the literature the majority of the pretended effects are mere statements in the prior art without any experimental support. We found on basis of a number of tests specifically developed in order to find experimental support to confirm the pretended effects that indeed some of the pretended effects exist however only to a low or medium extend.
Although WO 00/07607 discloses that a cancer-protective and cancer therapeutic composition is obtained by combining 1) a plant extract with antioxidant effect with 2) a neovascular regulator that inhibits angiogenesis and 3) with absorbable zinc whereas in the text the possibility of synergy between one or more of the components is suggested, there is no clear teaching that a synergy could be achieved by combining the components from which we found that they gave a synergy with respect to anti-inflammatory effects or with respect to skin benefits in particular to antiageing effects. In fact the preferred antioxidants are in this WO'607 F 7 551 cpl ( V ) CA 02354475 2001-08-O1 bioflavanoids such as proanthocyanidins. The neovascular regulator can be genistein, daidzein or a soy isolate.
We therefore studied whether we could find ways wherein the existing effects in particular with respect to anti-inflammatory and to antiageing of the skin of the isoflavones could be enhanced. As a result of this study we found that this can be done in a synergistic way by combining the effects from a number of selected isoflavones with the effect of a specific flavone. In fact we found that by combining one or more of the isoflavones selected from the group consisting of genistein, daidzein and glycetin with quercetin (= a specific flavone) synergistic effects could be achieved that were far higher than could be expected on basis of the components present in the combination. Therefore our invention concerns in the first instance a blend of a synergistic mix of a natural flavone and natural isoflavones, wherein the flavone is quercetin and the isoflavones are selected from at least one of the isoflavones from the group consisting of genestein, daidzein and glycetin either in the glucon or in the aglucon form.
In these blends the weight ratios quercetin to isoflavone can vary over a wide range, however we found that the best results were obtained when weight ratios of 1:50 to 50:1, preferably ratios of 1:20 to 20:1, more preferably ratios of 1:6 to 6:1 and even more preferably from 1:4 to 4:1 and most preferably from 1:2 to 2:1, calculated as aglucon, were applied. Even better results were obtained by using weight ratios of 1:2 to 1:1.
The most preferred isoflavones in these compositions are genestein and daidzein (or as glucons the genistin and daidzin). Although these components can be applied in a F 7 5 51 Cpl ( V ) CA 02354475 2001-08-O1 wide range of ratios the best results were obtained, when the genistein and daidzein were used in weight ratios of 2:1 to 1:2.
The isoflavones and the quercetin that can be applied according to the invention are suitably derived from natural sources. Quercetin e.g. is present in onions, garlic and tomatoes and concentrates wherein this component is present in relatively high levels can be obtained from these sources. Very suitable sources for the isoflavones are soy flour or clover and in particular extracts from soy or clover with an increased content of isoflavones, these concentrates are available as commercial products.
The application of above teaching might result in a blend wherein the quercetin is present in amounts of lOmg to 200 mg per RDI (recommended daily intake) while the isoflavones can be present in amounts of lOmg to 200mg per RDI. In this way the health components can be delivered as part of the daily servings of the food product.
According to another embodiment of our invention the invention also concerns food products containing a health component (= Functional food) wherein the food product comprises an amount of the blend of quercetin and at least one of the isoflavones genestein, daidzein and glycitin according to the invention, so that the total recommended daily intake (= RDI ) of the health components is delivered by one to 5 servings per day of the food product.
Typically the food products can be selected from the group consisting of spreads, margarines, creams, sauces, dressings, mayonnaises, ice creams, fillings, confectioneries, health bars, cereals, health drinks.
F 7 551 cpl ( V ) CA 02354475 2001-08-O1 In these food products 20 to 400 mg recommended daily intake of the synergistic blends according to the invention can be present. Because of the occurence of the synergy the food product can contain less of the individual flavone and iso-flavones, than otherwise would be required to achieve similar effects. In this way the performance of the food product is not negatively affected by the presence of the synergistic blend of health components while the health benefits are obtained.
In addition to the above components the blends and the food products can contain other micronutrients, examples thereof being anti oxidants (Vitamin C or Vitamin E), other vitamins in particular Vitamin B1, B6 and B12, Vitamin K, folic acid, minerals like calcium, magnesium, iron, copper, or zinc, however, emulsifiers also can be present as well as minor amounts of polyunsaturated fatty acids in particular DHA and EPA and in particular (deodorised) fish oils or concentrates thereof.
According to a last embodiment of our novel finding we also found that the blends or food products containing them can be used to achieve certain health effects, in particular certain cosmetical effects. Therefore our invention also concerns the use of a health composition comprising natural flavones and isoflavones wherein the health composition is the blend according to the invention or the food composition according to the invention and wherein the blend or food product is applied to achieve cosmetical effects, in particular skin benefits and skin related effects such as anti-ageing effects or for promoting the formation of collagen or for promoting the decorin formation in the skin. Further the invention concerns the use of a health composition comprising natural flavones and F 7551 Cpl (v) CA 02354475 2001-08-O1 isoflavones wherein the health composition is the blend according to the invention and wherein the blend is applied for the production of a functional food with anti-inflammatory properties with a synergistic effect on the 5 anti-inflammatory and related health properties.
This use can also result in a method for the treatment respectively the prevention of inflammations and related health deficiencies in animals or humans by administering to the animal or human in one or more servings in total an effective amount of the synergistic blend according to the invention or of the food product containing this blend according to the invention.
However the method can also comprise a method to achieve skin benefits or skin related effects, respectively to achieve the promotion of collagen formation or decorin formation in the skin by administering to an animal or human in one or more servings per day in total an effective amount of the synergistic blend according to the invention or the food product according to the invention.
In these methods for administering the amount to be administered should be the effective amount of the health component corresponding with the recommended daily intake of the isoflavones cq the flavone.
Procedure For Measuring Procollaqen-I and Decorin Synthesis In Human Dermal Fibroblasts Preparation of Dermal Fibroblast Conditioned Medium Primary human foreskin fibroblasts at passage 2 (P2) were seeded into 12-well plates at 10,000 cells/cm2 and maintained for 24 hours in atmospheric oxygen in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10~ foetal calf serum.
F 7 551 Cpl ( V ) CA 02354475 2001-08-O1 After this time the cells were washed with serum free DMEM
and then incubated in fresh serum free DMEM for a further 60 hours. Novasoy 40R containing 20 mM isoflavones, genistein, daidzein and glycetin (in a ratio of 1:1.3:0.3) and 5mM quercetin were, either independently or in combination added to the cells in triplicate in a final volume of 0.4m1/well fresh serum free DMEM and incubated for a further 24 hours. 1% ethanol was used as the vehicle control. This fibroblast conditioned medium was either analysed immediately or snap frozen in liquid nitrogen and stored at -70°C for future analysis. The cells were then counted and data from the dot-blot analysis subsequently standardised to cell number.
Dot Blot Assay for ProcollaQen-I and Decorin Protein in Dermal Fibroblast Conditioned Medium Samples of conditioned medium from dermal fibroblasts treated with actives as listed above were supplemented with 20mM dithiothreitol (1:10 dilution of 200mM stock solution) and 0.1~ sodium dodecylsulphate (1:100 dilution of 10°s stock solution), mixed well and then incubated at 75°C for 2 minutes. A standard for the assay was generated by serial dilution of neat fibroblast conditioned medium from fibroblasts seeded at 10,000 cells/cm2 in a 175 cm2 flask and maintained in serum free DMEM as described above.
Assay samples were subsequently applied in triplicate to a pre-wetted sheet of Immobilon-P transfer membrane using the 96-well Bio-Dot Apparatus from Bio-Rad as described in the manufacturers guidelines. Approximately 200u1 of medium was applied per well. The medium was allowed to filter through the membrane under gravity (30 minutes) after which the ~ 5 51 Cpl ( V ) CA 02354475 2001-08-O1 membrane was washed twice with PBS (200u1). These PBS
washes were allowed to filter through the membrane under gravity (2x15 minutes). The Bio-Dot apparatus was then attached to a vacuum manifold and a third and final PBS
wash carried out under suction. The apparatus was disassembled, the membrane removed and quickly cut as required before being placed in blocking buffer overnight at 4°C.
Membranes prepared for procollagen-I and decorin analysis were both blocked with 5% (w/v) non fat dried milk powder/
0.05s Tween 20 in PBS. The following day, the membranes were probed with 1:10000 dilution of primary antibodies to either human procollagen-I (MAB1912; rat monoclonal;
Chemicon Int. Inc., Temecula, CA) or human decorin (mouse polyclonal; AMS, UK) for 2 hours at room temperature. The membranes were subsequently washed with TBS/ 0.05°s Tween 20 (3 x 5 minutes) and then incubated with 1:1000 dilution of 125I-conjugated anti-rat or anti-mouse F(ab')2 fragments (ICN, Amersham respectively) as required for 1 hour at room temperature.
Following this the Immobilon strips were again washed with TBS/Tween 20 (3 x 5 minutes) before being allowed to dry in air at room temperature. The dried membranes were wrapped in cellophane and exposed to a Molecular Dynamics storage phosphor screen for 16-18 hours. At the end of this time the exposed screen was scanned by a phosphorimager (Molecular Dynamics Phophorimager SF) using ImageQuantTM
software. Dot intensity was assessed by computer-assisted image analysis using quantification tools in ImageQuantTM, standardised to cell number and the effects of various test reagents on decorin and procollagen-I synthesis were / 551 Cpl ~ V ~ CA 02354475 2001-08-O1 determined relative to a vehicle treated control value of 100 arbitrary units.
Results When quercetin and Novasoy 40 are added to cells at sub-optimal concentrations (i.e. those concentrations just below the concentrations that produce maximal upregulation of procollagen-1) the synthesis of procollagen-1 can be enhanced when quercetin and Novasoy 40 are combined at concentrations of 5uM and 20uM (isoflavones), respectively.
A synergistic interaction was observed between Novasoy 40 and quercetin when they were combined at the above concentrations. (cf. fig. l) Fibroblasts PGE2 Assay PGE2 production by human skin fibroblasts can be induced by the inflammatory stimulus PMA (phorbal myristate acetate).
PMA represents and external stressor which induces oxidative stress and inflammatory responses in cells. This model is used to model inflammation in vivo.
Primary human foreskin fibroblasts at passage 2 (P2) were seeded into 96-well plates at 35,000 cells/well and maintained for 24 hours in an atmosphere of 5% carbon dioxide in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal calf serum. Novasoy 40 R
containing 20 mM isoflavones, genistein, daidzein and glycetin (in a ratio of 1:1.3:0.3) and 5mM quercetin were , either independently or in combination added to the cells (DMEM, supplemented with 10% foetal calf serum) in dimethylsulphoxide (ethanol, final concentration 1%) in r~ / 551 Cpl ~ V ~ CA 02354475 2001-08-O1 triplicate and incubated for a further 24 hours. Phorbal myristate acetate (PMA) (Sigma) was added to the media and the cells incubated for a further 24 hours. The control did not contain any test compounds nor any PMA. The fibroblasts/media were then analysed immediately or snap frozen in liquid nitrogen and stored at -70°C for future analysis. The cells were then counted and data from the dot-blot analysis subsequently standardised to cell number.
Prostaglandin E2 (PGE2) assay: Volumes of 50u1 culture medium were taken for PGE2 assay after gently shaking the culture plate. PGE2 levels in the medium were determined with a Biotrak PGEZ immunoassay kit (Amersham, UK). The assay is based on the competition between unlabelled PGE2 in the sample and a fixed quantity of horseradish peroxidase labeled PGEZ for a limited amount of fixed PGE2 specific antibody. Concentrations of unlabeled sample PGEZ
are determined according to a standard curve, which was obtained at the same time.
When quercetin and Novasoy 40 are added to cells at sub-optimal concentrations (i.e. those concentrations just below the concentrations that produce maximal down-regulation of PGE2) the down-regulation of PGE2 can be enhanced when quercetin and Novasoy 40 are combined at concentrations of lOuM and 20uM (isoflavones), respectively. A synergistic interaction was observed between Novasoy 40 and quercetin when they were combined at the above concentrations. (cf. fig. 2) / 551 Cpl- ~ V ~ CA 02354475 2001-08-O1 Example RECIPE
3.4 grams of vegetable fat 0.5 g of modified egg yolk } together named "creamer"
6.0 g of maltodextrin 0.025 grams of Novosoy 40 R containing 40 wt of the o 10isoflavones genistin / daidzin and glycitin in a weight ratio of 1.3 . 1 . 0.3 0.010 grams of quercitin 0.6 grams of maize starch croutons 16.1 .grams of dried potato starch 151Øgrams of salt 0.3 grams of onion solids 0.7 grams of onions 0.2 grams of parsley and herb extract 3.2 grams of flavouring agents The creamer and the other components are mixed in mixer The blend obtained is a dried instant oninon soup hat can be t used for making a soup by mixing it with 200 of boiling ml water under stirring. The soup that is made thisway tastes 25very well.
Isoflavones are known as health components that can be applied to prevent or treat many health deficiencies or to achieve certain health effects not directly related with a health deficiency. E.g. these compounds are known to achieve benefits in the women's health area in particular for postmenopausal women. These effects are disclosed in e.g. US 5 498 631; WO 98/ 56373; WO 98/08503; US 5 733 926;
US 5 952 374 and many other references. Health effects that are also attributed to isoflavones include skin effects and anti-inflammatory effects.
Although for a few of these effects some experimental support can be found in the literature the majority of the pretended effects are mere statements in the prior art without any experimental support. We found on basis of a number of tests specifically developed in order to find experimental support to confirm the pretended effects that indeed some of the pretended effects exist however only to a low or medium extend.
Although WO 00/07607 discloses that a cancer-protective and cancer therapeutic composition is obtained by combining 1) a plant extract with antioxidant effect with 2) a neovascular regulator that inhibits angiogenesis and 3) with absorbable zinc whereas in the text the possibility of synergy between one or more of the components is suggested, there is no clear teaching that a synergy could be achieved by combining the components from which we found that they gave a synergy with respect to anti-inflammatory effects or with respect to skin benefits in particular to antiageing effects. In fact the preferred antioxidants are in this WO'607 F 7 551 cpl ( V ) CA 02354475 2001-08-O1 bioflavanoids such as proanthocyanidins. The neovascular regulator can be genistein, daidzein or a soy isolate.
We therefore studied whether we could find ways wherein the existing effects in particular with respect to anti-inflammatory and to antiageing of the skin of the isoflavones could be enhanced. As a result of this study we found that this can be done in a synergistic way by combining the effects from a number of selected isoflavones with the effect of a specific flavone. In fact we found that by combining one or more of the isoflavones selected from the group consisting of genistein, daidzein and glycetin with quercetin (= a specific flavone) synergistic effects could be achieved that were far higher than could be expected on basis of the components present in the combination. Therefore our invention concerns in the first instance a blend of a synergistic mix of a natural flavone and natural isoflavones, wherein the flavone is quercetin and the isoflavones are selected from at least one of the isoflavones from the group consisting of genestein, daidzein and glycetin either in the glucon or in the aglucon form.
In these blends the weight ratios quercetin to isoflavone can vary over a wide range, however we found that the best results were obtained when weight ratios of 1:50 to 50:1, preferably ratios of 1:20 to 20:1, more preferably ratios of 1:6 to 6:1 and even more preferably from 1:4 to 4:1 and most preferably from 1:2 to 2:1, calculated as aglucon, were applied. Even better results were obtained by using weight ratios of 1:2 to 1:1.
The most preferred isoflavones in these compositions are genestein and daidzein (or as glucons the genistin and daidzin). Although these components can be applied in a F 7 5 51 Cpl ( V ) CA 02354475 2001-08-O1 wide range of ratios the best results were obtained, when the genistein and daidzein were used in weight ratios of 2:1 to 1:2.
The isoflavones and the quercetin that can be applied according to the invention are suitably derived from natural sources. Quercetin e.g. is present in onions, garlic and tomatoes and concentrates wherein this component is present in relatively high levels can be obtained from these sources. Very suitable sources for the isoflavones are soy flour or clover and in particular extracts from soy or clover with an increased content of isoflavones, these concentrates are available as commercial products.
The application of above teaching might result in a blend wherein the quercetin is present in amounts of lOmg to 200 mg per RDI (recommended daily intake) while the isoflavones can be present in amounts of lOmg to 200mg per RDI. In this way the health components can be delivered as part of the daily servings of the food product.
According to another embodiment of our invention the invention also concerns food products containing a health component (= Functional food) wherein the food product comprises an amount of the blend of quercetin and at least one of the isoflavones genestein, daidzein and glycitin according to the invention, so that the total recommended daily intake (= RDI ) of the health components is delivered by one to 5 servings per day of the food product.
Typically the food products can be selected from the group consisting of spreads, margarines, creams, sauces, dressings, mayonnaises, ice creams, fillings, confectioneries, health bars, cereals, health drinks.
F 7 551 cpl ( V ) CA 02354475 2001-08-O1 In these food products 20 to 400 mg recommended daily intake of the synergistic blends according to the invention can be present. Because of the occurence of the synergy the food product can contain less of the individual flavone and iso-flavones, than otherwise would be required to achieve similar effects. In this way the performance of the food product is not negatively affected by the presence of the synergistic blend of health components while the health benefits are obtained.
In addition to the above components the blends and the food products can contain other micronutrients, examples thereof being anti oxidants (Vitamin C or Vitamin E), other vitamins in particular Vitamin B1, B6 and B12, Vitamin K, folic acid, minerals like calcium, magnesium, iron, copper, or zinc, however, emulsifiers also can be present as well as minor amounts of polyunsaturated fatty acids in particular DHA and EPA and in particular (deodorised) fish oils or concentrates thereof.
According to a last embodiment of our novel finding we also found that the blends or food products containing them can be used to achieve certain health effects, in particular certain cosmetical effects. Therefore our invention also concerns the use of a health composition comprising natural flavones and isoflavones wherein the health composition is the blend according to the invention or the food composition according to the invention and wherein the blend or food product is applied to achieve cosmetical effects, in particular skin benefits and skin related effects such as anti-ageing effects or for promoting the formation of collagen or for promoting the decorin formation in the skin. Further the invention concerns the use of a health composition comprising natural flavones and F 7551 Cpl (v) CA 02354475 2001-08-O1 isoflavones wherein the health composition is the blend according to the invention and wherein the blend is applied for the production of a functional food with anti-inflammatory properties with a synergistic effect on the 5 anti-inflammatory and related health properties.
This use can also result in a method for the treatment respectively the prevention of inflammations and related health deficiencies in animals or humans by administering to the animal or human in one or more servings in total an effective amount of the synergistic blend according to the invention or of the food product containing this blend according to the invention.
However the method can also comprise a method to achieve skin benefits or skin related effects, respectively to achieve the promotion of collagen formation or decorin formation in the skin by administering to an animal or human in one or more servings per day in total an effective amount of the synergistic blend according to the invention or the food product according to the invention.
In these methods for administering the amount to be administered should be the effective amount of the health component corresponding with the recommended daily intake of the isoflavones cq the flavone.
Procedure For Measuring Procollaqen-I and Decorin Synthesis In Human Dermal Fibroblasts Preparation of Dermal Fibroblast Conditioned Medium Primary human foreskin fibroblasts at passage 2 (P2) were seeded into 12-well plates at 10,000 cells/cm2 and maintained for 24 hours in atmospheric oxygen in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10~ foetal calf serum.
F 7 551 Cpl ( V ) CA 02354475 2001-08-O1 After this time the cells were washed with serum free DMEM
and then incubated in fresh serum free DMEM for a further 60 hours. Novasoy 40R containing 20 mM isoflavones, genistein, daidzein and glycetin (in a ratio of 1:1.3:0.3) and 5mM quercetin were, either independently or in combination added to the cells in triplicate in a final volume of 0.4m1/well fresh serum free DMEM and incubated for a further 24 hours. 1% ethanol was used as the vehicle control. This fibroblast conditioned medium was either analysed immediately or snap frozen in liquid nitrogen and stored at -70°C for future analysis. The cells were then counted and data from the dot-blot analysis subsequently standardised to cell number.
Dot Blot Assay for ProcollaQen-I and Decorin Protein in Dermal Fibroblast Conditioned Medium Samples of conditioned medium from dermal fibroblasts treated with actives as listed above were supplemented with 20mM dithiothreitol (1:10 dilution of 200mM stock solution) and 0.1~ sodium dodecylsulphate (1:100 dilution of 10°s stock solution), mixed well and then incubated at 75°C for 2 minutes. A standard for the assay was generated by serial dilution of neat fibroblast conditioned medium from fibroblasts seeded at 10,000 cells/cm2 in a 175 cm2 flask and maintained in serum free DMEM as described above.
Assay samples were subsequently applied in triplicate to a pre-wetted sheet of Immobilon-P transfer membrane using the 96-well Bio-Dot Apparatus from Bio-Rad as described in the manufacturers guidelines. Approximately 200u1 of medium was applied per well. The medium was allowed to filter through the membrane under gravity (30 minutes) after which the ~ 5 51 Cpl ( V ) CA 02354475 2001-08-O1 membrane was washed twice with PBS (200u1). These PBS
washes were allowed to filter through the membrane under gravity (2x15 minutes). The Bio-Dot apparatus was then attached to a vacuum manifold and a third and final PBS
wash carried out under suction. The apparatus was disassembled, the membrane removed and quickly cut as required before being placed in blocking buffer overnight at 4°C.
Membranes prepared for procollagen-I and decorin analysis were both blocked with 5% (w/v) non fat dried milk powder/
0.05s Tween 20 in PBS. The following day, the membranes were probed with 1:10000 dilution of primary antibodies to either human procollagen-I (MAB1912; rat monoclonal;
Chemicon Int. Inc., Temecula, CA) or human decorin (mouse polyclonal; AMS, UK) for 2 hours at room temperature. The membranes were subsequently washed with TBS/ 0.05°s Tween 20 (3 x 5 minutes) and then incubated with 1:1000 dilution of 125I-conjugated anti-rat or anti-mouse F(ab')2 fragments (ICN, Amersham respectively) as required for 1 hour at room temperature.
Following this the Immobilon strips were again washed with TBS/Tween 20 (3 x 5 minutes) before being allowed to dry in air at room temperature. The dried membranes were wrapped in cellophane and exposed to a Molecular Dynamics storage phosphor screen for 16-18 hours. At the end of this time the exposed screen was scanned by a phosphorimager (Molecular Dynamics Phophorimager SF) using ImageQuantTM
software. Dot intensity was assessed by computer-assisted image analysis using quantification tools in ImageQuantTM, standardised to cell number and the effects of various test reagents on decorin and procollagen-I synthesis were / 551 Cpl ~ V ~ CA 02354475 2001-08-O1 determined relative to a vehicle treated control value of 100 arbitrary units.
Results When quercetin and Novasoy 40 are added to cells at sub-optimal concentrations (i.e. those concentrations just below the concentrations that produce maximal upregulation of procollagen-1) the synthesis of procollagen-1 can be enhanced when quercetin and Novasoy 40 are combined at concentrations of 5uM and 20uM (isoflavones), respectively.
A synergistic interaction was observed between Novasoy 40 and quercetin when they were combined at the above concentrations. (cf. fig. l) Fibroblasts PGE2 Assay PGE2 production by human skin fibroblasts can be induced by the inflammatory stimulus PMA (phorbal myristate acetate).
PMA represents and external stressor which induces oxidative stress and inflammatory responses in cells. This model is used to model inflammation in vivo.
Primary human foreskin fibroblasts at passage 2 (P2) were seeded into 96-well plates at 35,000 cells/well and maintained for 24 hours in an atmosphere of 5% carbon dioxide in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal calf serum. Novasoy 40 R
containing 20 mM isoflavones, genistein, daidzein and glycetin (in a ratio of 1:1.3:0.3) and 5mM quercetin were , either independently or in combination added to the cells (DMEM, supplemented with 10% foetal calf serum) in dimethylsulphoxide (ethanol, final concentration 1%) in r~ / 551 Cpl ~ V ~ CA 02354475 2001-08-O1 triplicate and incubated for a further 24 hours. Phorbal myristate acetate (PMA) (Sigma) was added to the media and the cells incubated for a further 24 hours. The control did not contain any test compounds nor any PMA. The fibroblasts/media were then analysed immediately or snap frozen in liquid nitrogen and stored at -70°C for future analysis. The cells were then counted and data from the dot-blot analysis subsequently standardised to cell number.
Prostaglandin E2 (PGE2) assay: Volumes of 50u1 culture medium were taken for PGE2 assay after gently shaking the culture plate. PGE2 levels in the medium were determined with a Biotrak PGEZ immunoassay kit (Amersham, UK). The assay is based on the competition between unlabelled PGE2 in the sample and a fixed quantity of horseradish peroxidase labeled PGEZ for a limited amount of fixed PGE2 specific antibody. Concentrations of unlabeled sample PGEZ
are determined according to a standard curve, which was obtained at the same time.
When quercetin and Novasoy 40 are added to cells at sub-optimal concentrations (i.e. those concentrations just below the concentrations that produce maximal down-regulation of PGE2) the down-regulation of PGE2 can be enhanced when quercetin and Novasoy 40 are combined at concentrations of lOuM and 20uM (isoflavones), respectively. A synergistic interaction was observed between Novasoy 40 and quercetin when they were combined at the above concentrations. (cf. fig. 2) / 551 Cpl- ~ V ~ CA 02354475 2001-08-O1 Example RECIPE
3.4 grams of vegetable fat 0.5 g of modified egg yolk } together named "creamer"
6.0 g of maltodextrin 0.025 grams of Novosoy 40 R containing 40 wt of the o 10isoflavones genistin / daidzin and glycitin in a weight ratio of 1.3 . 1 . 0.3 0.010 grams of quercitin 0.6 grams of maize starch croutons 16.1 .grams of dried potato starch 151Øgrams of salt 0.3 grams of onion solids 0.7 grams of onions 0.2 grams of parsley and herb extract 3.2 grams of flavouring agents The creamer and the other components are mixed in mixer The blend obtained is a dried instant oninon soup hat can be t used for making a soup by mixing it with 200 of boiling ml water under stirring. The soup that is made thisway tastes 25very well.
Claims (10)
1. A blend of a synergistic mix of a natural flavone and natural isoflavones, wherein the flavone is quercetin and the isoflavones are selected from at least one of isoflavone, selected from the group consisting of genestein, daidzein and glycetin, either in the glucon or in the aglucon form, and wherein quercetin and the natural isoflavone are present in weight ratios of 1:50 to 50:1, calculated as aglucon, while genestein and daidzein are present in a weight ratio (as aglucon) of 2:1 to 1:2.
2. The blend according to claim 1, wherein quercetin and the natural isoflavone are present in a weight ratio of from 1:20 to 20:1, calculated as aglucon.
3. The blend according to claim 1, wherein quercetin and the natural isoflavone are present in weight ratios of 1:6 to 6:1, calculated as aglucon.
4. The blend according to claim 1, wherein quercetin and the natural isoflavone are present in a weight ratio of 1:4 to 4:1, calculated as aglucon.
5. The blend according to any one of claims 1 to 4, wherein the weight ratio of quercetin to total isoflavones is in the range of from 1:2 to 2:1.
6. The blend according to any one of claims 1 to 5, wherein the weight ratio of quercetin to genestein, as aglucon, is in the range of from 1:2 to 1:1.
7. The blend according to any one of claims 1 to 6, wherein the natural isoflavones are derived from soy and comprise soy flour or soy extracts.
8. The blend according to any one of claims 1 to 7, wherein the quercetin is a natural product derived from onion or from garlic or from tomatoes.
9. The blend according to claim 8, wherein said natural product is a concentrate of onions, or of garlic or of tomatoes.
10. A food product comprising the blend of quercetin and at least one of the isoflavones genestein, daidzein and glycitin according to any one of claims 1 to 9, wherein the food product is selected from the group consisting of spreads, margarines, creams, sauces, dressings, mayonnaises, ice creams, fillings, confectioneries, health bars, cereals and health drinks.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP00307031 | 2000-08-16 | ||
| EP00307031.5 | 2000-08-16 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2354475A1 CA2354475A1 (en) | 2002-02-16 |
| CA2354475C true CA2354475C (en) | 2011-09-20 |
Family
ID=8173193
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2354475A Expired - Fee Related CA2354475C (en) | 2000-08-16 | 2001-08-01 | Blends of isoflavones and flavones |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US6617349B2 (en) |
| AT (1) | ATE400285T1 (en) |
| AU (1) | AU5780901A (en) |
| CA (1) | CA2354475C (en) |
| DE (1) | DE60134702D1 (en) |
| ES (1) | ES2309030T3 (en) |
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|---|---|---|---|---|
| US7338971B2 (en) * | 2001-08-30 | 2008-03-04 | El-Naggar Mawaheb M | Treatment of inflammatory, cancer, and thrombosis disorders |
| US20030144219A1 (en) * | 2001-11-15 | 2003-07-31 | Phinney Stephen Dodge | Formulations and methods for treatment or amelioration of inflammatory conditions |
| WO2004004638A2 (en) * | 2002-07-02 | 2004-01-15 | Galileo Pharmaceuticals, Inc. | Compositions and methods for reduction of inflammatory symptoms and/or biomarkers in female subjects |
| GB0306568D0 (en) * | 2003-03-21 | 2003-04-30 | Unilever Plc | Compositions of natural products and use thereof |
| EP1691759A4 (en) * | 2003-11-13 | 2008-08-06 | Johnson & Johnson Consumer | Plant-derived protein extract compositions and methods |
| US20060188590A1 (en) * | 2005-01-05 | 2006-08-24 | Mitsunori Ono | Compositions for treating diabetes or obesity |
| US7871609B2 (en) * | 2007-03-02 | 2011-01-18 | Sam Ziff | Supplements for pain management |
| EP2365807B1 (en) * | 2008-12-11 | 2017-07-05 | Unilever PLC | Oral composition comprising daidzein and an anthocyanidin |
| CN109043526A (en) * | 2018-09-10 | 2018-12-21 | 漳州何氏农业开发有限公司 | A kind of beautifying face and moisturizing skin food and preparation method thereof |
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|---|---|---|---|---|
| AU6635994A (en) | 1993-04-16 | 1994-11-08 | Tufts University School Of Medicine | Method for treatment of menopausal and premenstrual symptoms |
| AUPO203996A0 (en) | 1996-08-30 | 1996-09-26 | Novogen Research Pty Ltd | Therapeutic uses |
| US5733926A (en) | 1996-12-13 | 1998-03-31 | Gorbach; Sherwood L. | Isoflavonoids for treatment and prevention of alzheimer dementia and reduced cognitive functions |
| US6060070A (en) | 1997-06-11 | 2000-05-09 | Gorbach; Sherwood L. | Isoflavonoids for treatment and prevention of aging skin and wrinkles |
| US5952374A (en) | 1997-09-29 | 1999-09-14 | Protein Technologies International, Inc. | Method for inhibiting the development of Alzheimer's disease and related dementias- and for preserving cognitive function |
| MXPA00012844A (en) | 1998-07-07 | 2004-05-21 | Transdermal Technologies Inc | Compositions for rapid and non-irritating transdermal delivery of pharmaceutically active agents and methods for formulating such compositions and delivery thereof. |
| AU5334899A (en) | 1998-08-04 | 2000-02-28 | Kosbab, John V. | Nutrient and therapeutic compositions for the treatment of cancer |
| US6194469B1 (en) | 1998-12-11 | 2001-02-27 | Board Of Trustees Operating Michigan State Univeristy | Method for inhibiting cyclooxygenase and inflammation using cherry bioflavonoids |
| MXPA00012314A (en) | 1999-04-08 | 2002-10-23 | Metagenics Inc | Composition and method for treatment of inflammation and pain in mammals. |
| US20020160060A1 (en) | 1999-04-26 | 2002-10-31 | Mandy Kim Chen | Enriched spreads |
| US6210701B1 (en) * | 1999-04-30 | 2001-04-03 | Healthcomm International, Inc. | Medical food for treating inflammation-related diseases |
| DE10009424A1 (en) | 2000-02-28 | 2001-09-06 | Henkel Kgaa | Use of flavone and isoflavone compounds, especially of plant origin, for the treatment of cellulite |
-
2001
- 2001-07-20 ES ES01202796T patent/ES2309030T3/en not_active Expired - Lifetime
- 2001-07-20 DE DE60134702T patent/DE60134702D1/en not_active Expired - Lifetime
- 2001-07-20 AT AT01202796T patent/ATE400285T1/en not_active IP Right Cessation
- 2001-08-01 CA CA2354475A patent/CA2354475C/en not_active Expired - Fee Related
- 2001-08-07 AU AU57809/01A patent/AU5780901A/en not_active Abandoned
- 2001-08-15 US US09/930,525 patent/US6617349B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| ES2309030T3 (en) | 2008-12-16 |
| DE60134702D1 (en) | 2008-08-21 |
| AU5780901A (en) | 2002-02-21 |
| ATE400285T1 (en) | 2008-07-15 |
| US20020068121A1 (en) | 2002-06-06 |
| US6617349B2 (en) | 2003-09-09 |
| CA2354475A1 (en) | 2002-02-16 |
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